Central to Q fever pathogenesis is replication of the causative agent, Coxiella burnetii, in a large and spacious phagolysosome-like parasitophorous vacuole (PV). The recruitment of membrane during PV biogenesis is a complex process that is modulated by both host and bacterial factors. Moreover, the lipid content of the PV membrane may confer unique properties that allow pathogen growth. Cholesterol is a major sterol component of mammalian membranes where it provides structural stability, signaling platforms called lipid rafts, and serves as a precursor of secondary messenger molecules. Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in Coxiella infection utilizing DHCR24-/- mouse embryonic fibroblasts (MEFs). DHCR24-/- MEFs lack the delta24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24-/- MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for Coxiella with Salmonella enterica serovar Typhimurium and Chalmydia trachomatis employed as control organisms. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24-/- MEFs. In contrast, Coxiella entry, but not attachment, was significantly decreased, suggesting Coxiella utilizes lipid-raft mediated signaling to gain entry into host cells. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the Coxiella-occupied vacuole within DHCR24-/- MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, suggesting cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data indicate cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in Coxiella-host cell interactions. Coxiella encodes a specialized Dot/Icm Type IVB secretion system (T4BSS) that secretes proteins with effector functions directly into the host cell cytosol. Effector proteins are predicted to modulate an array of host cell processes, such as vesicular trafficking, that promote pathogen growth. Coxiella Dot/Icm function was initially studied using Legionella pneumophila as surrogate host. However, by using new gene inactivation technologies developed in our laboratory, we have recently confirmed that a functional T4BSS is required for productive infection of human macrophages by Coxiella. Furthermore, we have verified Dot/Icm-dependent secretion by Coxiella of over 30 proteins. Coxiella must co-opt vesicular trafficking pathways to promote PV development. We are currently elucidating the activities of four proteins that traffic to the PV membrane when ectopically expressed in infected cells termed CvpA (Coxiella vacuolar protein A), CvpB, CvpC, and CvpD that are speculated to modulate membrane fusion events. Particular insight into the function of CvpA has been grained. A Coxiella cvpA mutant exhibits significant defects in replication and PV development. CvpA contains multiple dileucine DERQXXXLL,I and tyrosine (YXX&#934;)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. Ectopically expressed mCherry-CvpA localizes to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions are lost upon mutation of endocytic sorting motifs. In pull-down assays, peptides containing CvpA sorting motifs and full-length CvpA interact with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibits Coxiella replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in Coxiella infection and define a novel role for CvpA in subverting these transport mechanisms. Although T4BSS delivery of proteins into the host cell cytoplasm is clearly required for productive infection by Coxiella, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promotes pathogen replication. To assess the potential of Coxiella to secrete proteins into the PV, we analyzed by mass spectrometry the protein content of axenic growth media for the presence of pathogen proteins. From a candidate list of 55 identified proteins, secretion of 27 was confirmed by expressing FLAG-tagged proteins in Coxiella followed by immunoblotting of culture supernatants. Tagged proteins expressed by Coxiella transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs in vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase. Possible roles for secreted proteins based on genome annotation include detoxification of reactive oxygen species, transport of arginine, and degradation of protein. We propose that the majority of the sec-dependent secretome results from release of outer membrane vesicles (OMV). This idea is supported by EM showing obvious membrane blebbing and OMV production during growth of Coxiella in media and within mammalian host cells. An intracellular biphasic developmental cycle where resistant small cell variant (SCV) morphological forms are generated from large cell variant (LCV) morphological forms is considered fundamental to Coxiella virulence. However, the molecular biology of Coxiella development is poorly understood. Because intracellular growth of Coxiella imposes considerable experimental constraints, we sought to establish whether Coxiella developmental transitions in host cells are recapitulated during host cell-free (axenic) growth in first and second generation acidified citrate cysteine media (ACCM-1 and ACCM-2, respectively). We show that ACCM-2 supports developmental transitions and viability. Although ACCM-1 also supported SCV to LCV transition, LCV to SCV transition did not occur after extended incubation (21 days). Instead, Coxiella exhibited a ghost-like appearance with bacteria containing condensed chromatin but otherwise devoid of cytoplasmic content. This phenotype correlated with a near total loss in viability between 14 and 21 days of cultivation. Transcriptional profiling of Coxiella following 14 days of incubation revealed elevated expression of oxidative stress genes in ACCM-1 cultivated bacteria. The only difference between ACCM-1 and ACCM-2 is the substitution of fetal bovine serum for methyl-beta-cyclodextrin. Addition of methyl-beta-cyclodextrin to ACCM-1 at 7 days post-inoculation rescued Coxiella viability and lowered expression of oxidative stress genes. Thus, methyl-beta-cyclodextrin appears to alleviate oxidative stress in ACCM-2 to result in Coxiella developmental transitions and viability that mimic host cell-cultivated organisms. Axenic cultivation of Coxiella in ACCM-2, along with new methods for genetic manipulation, now provides powerful tools to investigate the molecular basis and biological relevance of Coxiella biphasic development.